Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD(+)-dependent Sir2 histone/protein deacetylases

Abstract

Sir2 enzymes are broadly conserved from bacteria to humans and have been implicated to play roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. These enzymes bind NAD(+) and acetyllysine within protein targets and generate lysine, 2'-O-acetyl-ADP-ribose, and nicotinamide products. To provide structural insights into the chemistry catalyzed by Sir2 proteins we report the high-resolution ternary structure of yeast Hst2 (homologue of Sir two 2) with an acetyllysine histone H4 peptide and a nonhydrolyzable NAD(+) analogue, carba-NAD(+), as well as an analogous ternary complex with a reaction intermediate analog formed immediately after nicotinamide hydrolysis, ADP-ribose. The ternary complex with carba-NAD(+) reveals that the nicotinamide group makes stabilizing interactions within a binding pocket harboring conserved Sir2 residues. Moreover, an asparagine residue, N116, strictly conserved within Sir2 proteins and shown to be essential for nicotinamide exchange, is in position to stabilize the oxocarbenium intermediate that has been proposed to proceed the hydrolysis of nicotinamide. A comparison of this structure with the ADP-ribose ternary complex and a previously reported ternary complex with the 2'-O-acetyl-ADP-ribose reaction product reveals that the ribose ring of the cofactor and the highly conserved beta1-alpha2 loop of the protein undergo significant structural rearrangements to facilitate the ordered NAD(+) reactions of nicotinamide cleavage and ADP-ribose transfer to acetate. Together, these studies provide insights into the chemistry of NAD(+) cleavage and acetylation by Sir2 proteins and have implications for the design of Sir2-specific regulatory molecules.

Fig. 1.

Overall structure of the yHst2 substrate and intermediate analog complexes. (a) Ribbon diagram of the yHst2/carba-NAD⁺/H4 complex highlighting the large domain (cyan), small domain (blue), and connecting loops (purple). The NAD⁺ (yellow), histone H4 peptide (green), and Zn ion (red) are also highlighted. (b) Superimposition of yHst2/carba-NAD⁺/H4 (cyan), yHst2/ADP-ribose/H4 (blue), and yHst2/2′-O-acetyl-ADP-ribose/H4 (purple). (c) Molecular surface of yHst2, highlighting the conformational difference of the β1–α2 NAD⁺-binding loops in the yHst2/carba-NAD⁺/H4 (cyan) and yHst2/2′-O-acetyl-ADP-ribose/H4 (red) complexes.

Fig. 2.

yHst2-NAD analog interaction. (a) The carba-NAD⁺-binding site of yHst2 highlighting the yHst2 residues (carbon, gray; oxygen, red; and nitrogen, blue) that contact the nicotinamide and nicotinamide-ribose (carbon, yellow). A simulated annealing F_o – F_c omit map of carba-NAD⁺ is counted at 3.0 σ (cyan) and the acetyllysine substrate (green) is also highlighted. (b) Summary of yHst2 carba-NAD⁺ interactions. Hydrogen bonds are indicated with dashed lines and van der Waals interactions are indicated with half-moon symbols. The residues highlighted in cyan take part in conserved interactions with the yHst2/2′-O-acetyl-ADP-ribose and yHst2/ADP-ribose complexes. The residues colored in red take part in nonconserved interactions and the residues highlighted in blue take part in interactions with the nicotinamide group. (c) The ADP-ribose-binding site of yHst2, highlighting the yHst2 residues that contact the nicotinamide and nicotinamide-ribose. A simulated annealing F_o – F_c omit map of ADP-ribose counted at 3.0 σ and the color coding are as described for a. (d) Summary of yHst2–ADP-ribose interactions. The coding for interactions and color designations are as described for b. (e) Overlay of the carba-NAD⁺ (light blue), ADP-ribose (dark blue), and 2′-O-acetyl-ADP-ribose (yellow) ligands, using the corresponding proteins for the superpositions. The acetyl group from the acetyllysine substrate of the respective complex is also shown in the same color.

Fig. 3.

The yHst2 nicotinamide-binding site. (a) van der Waals surface of yHst2/carba-NAD⁺/H4 highlighting invariant residues (blue) as well as the subset of these residues that contact nicotinamide (red). I117, which contacts nicotinamide, is also highlighted in red, although this residue has not been subjected to mutagenesis. The surface for the β1–α2 NAD⁺-binding loop is deleted from this image for clarity. (b) Stereoview of yHst2–nicotinamide interactions, with hydrogen bonds depicted as dotted lines. Protein residues that mediate van der Waals interactions with the nicotinamide are also highlighted. yHst2 and carba-NAD⁺ color-coding is as in Fig. 2a.

Fig. 4.

Proposed catalytic mechanism for yHst2.

Fig. 5.

The β1–α2 loop of yHst2 and analog binding. (a) Stereoview of an overlay of the β1–α2 loops from the yHst2/carba-NAD⁺/H4 (cyan for loop and light blue for carba-NAD⁺) and yHst2/2′-O-acetyl-ADP-ribose/H4 (purple for loop and yellow for 2′-O-acetyl-ADP-ribose) complexes. Residues D43, F44, R45, and Y52 and the respective NAD⁺ analogues are also highlighted in each structure. (b and c) Representative data of isothermal titration calorimetry for ADP-ribose titrations into yHst2 (b) and binary yHst2/K16-acetylated histone H4 peptide (c). The area under each injection spike (above) is integrated and fitted by using nonlinear least-squares regression analysis (below). For yHst2 and the binary complex, respectively, the enthalpy of reaction (ΔH = –67.50 ± 0.19 kJ/mol and –17.97 ± 1.20 kJ/mol), dissociation constant (K_d = 0.404 ± 0.001 μM and 29.16 ± 4.50 μM) and stoichiometry (n = 1.114 ± 0.004 and 0.910 ± 0.001) is calculated from the regression analysis. (d) Model for transglycosidation by yHst2. Nicotinamide (red) was modeled onto the ADP-ribose structure (yellow) in a geometry that closely mimics the glycosidic bond geometry between the nicotinamide and ADP-ribose as observed in carba-NAD⁺ (black, overlaid).